LOCUS       sequence-455373-        6521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express node 412 retron with split retron ncRNA and gRNA to edit 
            (10bp barcode) EMX1 gene in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6521)
  AUTHORS   Khan AG, Rojas-Montero M, Gonzalez-Delgado A, Lopez SC, Fang RF, 
            Crawford KD, Shipman SL
  TITLE     An experimental census of retrons for DNA production and genome 
            editing.
  JOURNAL   Nat Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z.
  PUBMED    39289529
REFERENCE   2  (bases 1 to 6521)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z."
COMMENT     Sequence Label: pAGK56
FEATURES             Location/Qualifiers
     source          1..6521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        368..645
                     /label=chicken beta-actin promoter
     intron          646..1662
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1586..1608
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1670..1689
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2765..2785
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2813..3037
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3072..3312
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     3072..3092
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3243..3262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3652..3866
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3787..3806
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(4006..4022)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4006..4022)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4019..4041)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4030..4046
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4054..4060,4061..4078,4079..4084))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4099..4120
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4179..4374
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4225..4360
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4380..4514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4417..4436)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4471..4490
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4582..4599)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4753..5341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4833..4852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5512..6303,6304..6372))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6135..6154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6373..6477)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6508..6521,1..366)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
      421 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
      481 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
      541 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
      601 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      661 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      721 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      781 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
      841 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
      901 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
      961 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
     1021 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
     1081 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
     1141 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
     1201 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
     1261 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
     1321 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
     1381 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
     1441 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
     1501 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
     1561 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
     1621 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
     1681 gttattgtgc tgtctcatca ttttggcaaa gaattgagct cgatatcggt acgccaccat
     1741 gaattggaag aaataccgca aggagtttac catccaggct accgccaagt ctctggatga
     1801 gaagagcatg cagcacgccc tgagttatgc ccgcggcttg tacaaggcaa acctgccaat
     1861 tatcttttcc cagaaccacc tgagccgcct cgtgggctat aacatcgatt acatccgcaa
     1921 cgcttcttcc actaagtccg atcagttcta tagatgctac aagattaaaa agaaatcatc
     1981 cgatactatg cgcactatct gtgagccact gccgagcctg aaggagatcc agcgctggat
     2041 cctggacaac atcctgagcc aggtcacgat ctccaagttt gctaaggcct acgtcgtggg
     2101 attttctatc aaggacaacg caaggtttca cctgcgccag aaacaagtcc tccgcatcga
     2161 tgtcaaggac ttctttccaa gcatcaaggg ccagaacgtc tttcacatct ttaaaaatat
     2221 tggctattct gctgaggtga gcgcaatgct gaccggcctg acgaccctga agaactgcct
     2281 cccccagggt gcgcctactt ccccgactct gtccaacatc tttatgaacc gctgcgacgc
     2341 caggattgcc ggctactgtc tggcaaggaa gattcgttac acccgctaca gcgacgatct
     2401 gacgttcagc ggcgagttcg atgtcggtaa gctgatctct ttcatcaaca tggtgtttaa
     2461 ggactctggc cttctgttga acaagaccaa gacgaagcag atgtttaagc atcagcgcca
     2521 gttcacaacc ggtctggtcg tgaacgagag gatcaacgta tgtcgcgaga agcgtcgcaa
     2581 gctgcggcag gagatttttt acatcaaaaa gtacggcctc gacggacacc tgattaggac
     2641 cgaagagaag cgcacgaatt acatcatgca cttgcttggc ctgtccaatt tcatcacaca
     2701 catcaacgct aacgaccgcg atgcactgga ggcgaaggaa atcctcagga aatatattac
     2761 gccaccaaag aagaaaagaa aggtctgatc ccggttagta atgagtttaa acctgtgcct
     2821 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2881 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2941 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3001 aatagcaggc atgctgggga tgcggtgggc tctatggaga tctgcgaaat cggatccagc
     3061 tgaatctaag tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     3121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     3181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     3241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     3301 tgtggaaagg acgaaaccgt acggcctcac tagccttagt ggcttagcca cggaatggcg
     3361 agtcgccatt ctggggagaa cttgctttca agcccaggcg aagattcacc aactcttgga
     3421 aagagttggc gaatcttcgc cccgcttccg caaaagcctc cagccccgag atgacaaacg
     3481 gcagaagctg gaggaggaag ggcctgagtc cgagcagaag aagctcaatc agaaagttct
     3541 cccatcacat caaccggtgg cgcattgcca cgaagcaggc caatggggag gacatcgatg
     3601 tcacatctcg gtgattatgt ccgtatgtag tgaggccgta cggttttttt tgaacgctga
     3661 cgtcatcaac ccgctccaag gaatcgcggg cccagtgtca ctaggcggga acacccagcg
     3721 cgcgtgcgcc ctggcaggaa gatggctgtg agggacaggg gagtggcgcc ctgcaatatt
     3781 tgcatgtcgc tatgtgttct gggaaatcac cataaacgtg aaatgtcttt ggatttggga
     3841 atcttataag ttctgtatga gaccacgaaa gagtccgagc agaagaagaa gtttcagagc
     3901 tatgctggaa acagcatagc aagttgaaat aaggctagtc cgttatcaac ttgaaaaagt
     3961 ggcaccgagt cggtgctttt ttttttttag cttggcgtaa tcatggtcat agctgtttcc
     4021 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4081 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4141 cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc
     4201 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4261 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4321 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
     4381 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4441 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4501 atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4561 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4621 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4681 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4741 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4801 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4861 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4921 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4981 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5041 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5101 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5161 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5221 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5281 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5341 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5401 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5461 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5521 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5581 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5641 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5701 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5761 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5821 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5881 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5941 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6001 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6061 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6121 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6181 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6241 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6301 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6361 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6421 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6481 atttccccga aaagtgccac ctgggtcgac attgattatt g
//
